WO2014007190A1 - Cell-spreading device and method for detecting rare cell - Google Patents
Cell-spreading device and method for detecting rare cell Download PDFInfo
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- WO2014007190A1 WO2014007190A1 PCT/JP2013/067978 JP2013067978W WO2014007190A1 WO 2014007190 A1 WO2014007190 A1 WO 2014007190A1 JP 2013067978 W JP2013067978 W JP 2013067978W WO 2014007190 A1 WO2014007190 A1 WO 2014007190A1
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Definitions
- the present invention relates to a microchamber chip having a microchamber on the surface capable of storing and holding cells, a flow channel forming frame, and an inlet portion and an outlet portion of a cell suspension provided in the flow channel forming frame. And a method for detecting rare cells from a cell suspension using the device.
- CTC circulating tumor cell
- FDA US Food and Drug Administration
- CTC has a very low blood concentration
- high detection power is required to detect CTC.
- a detection region for example, an observation field of a microscope
- FIG. 7 shows a state in which red blood cells are stored in the microchamber in three types of microarray chips A to C each having a microchamber having a specific inner diameter and depth.
- the gaps between the micro chambers are large. Therefore, when a sample is simply added on the surface of the microarray chip, it can be easily estimated that a large amount of cells remain outside the microchamber.
- Other such microarray chips are disclosed in Patent Documents 2 and 3 and Non-Patent Document 1, respectively.
- microchamber type device in view of industrial mass productivity, molding (or production) using a mold is common.
- the microchambers are in close contact with each other, that is, the area outside the microchamber is minimized.
- Limited micro-chamber type devices are also conceivable, but it is not realistic to assume that the micro-chamber type device is "extracted" from the mold during mold production. it is obvious.
- the present invention develops a cell suspension on the surface of a microchamber chip when detecting rare cells (for example, CTC) from a cell suspension containing a large amount of various types of cells such as blood.
- CTC rare cells
- the ratio of the total number of cells that can be held in each microchamber relative to the total number of cells contained in the cell suspension (hereinafter also referred to as “cell recovery rate”) is extremely high.
- An object of the present invention is to provide a cell deployment device having a microchamber chip that can be produced.
- the present inventors have found that the cell suspension is on the surface of a microchamber chip on which the microchamber is arranged under specific conditions. When it was allowed to flow down, it was found that the cell recovery rate was extremely high, and the present invention was completed.
- the cell deployment device stores and holds cells according to, for example, FIG.
- a device for deployment (10), in which the opening of the microchamber (6) is vertically projected onto the vertical width (20) of the microchamber chip (1) Is the ratio of the sum of the voids (40), which is the length of the portion where the projected opening of the micro chamber (6) does not exist with respect to the vertical width (20), to the vertical width (20).
- the porosity is 5% or less.
- the cell expansion device When the cell expansion device according to the present invention is used, for example, when all cells contained in a cell suspension such as blood are expanded on a microchamber chip, the cells are almost discharged from the outlet of the cell expansion device. Almost all cells can be kept in the microchamber. Moreover, since such a thing can be performed after maintaining a fixed space
- the cells stored and retained in the microchamber may be retained in the microchamber even after the staining process or the washing process. Almost no leakage from
- FIG. 1 schematically shows a cross-sectional view parallel to the direction in which a cell suspension flows when the cell suspension flows through the flow path (5) of the cell deployment device (10) according to the present invention.
- FIG. 1 (A) shows an embodiment in which the bottom surface of the flow path (5) is made of the surface of the microchamber chip (1)
- FIG. 1 (B) shows that the bottom surface of the flow path is the micro chamber chip (1 ) And a part of the flow path forming frame (2).
- a dotted arrow indicates a direction in which the cell suspension flows when the cell suspension is introduced into the cell deployment device (10) according to the present invention.
- FIG. 1 schematically shows a cross-sectional view parallel to the direction in which a cell suspension flows when the cell suspension flows through the flow path (5) of the cell deployment device (10) according to the present invention.
- FIG. 1 (A) shows an embodiment in which the bottom surface of the flow path (5) is made of the surface of the microchamber chip (1)
- FIG. 1 (B) shows that the bottom surface of
- FIG. 2 is a schematic view of a part of one embodiment of the microchamber chip (1) used in the present invention as seen from directly above, and shows the vertical width (20) and the gap (40) of the microchamber chip (1). Show the relationship. Note that the direction indicated by the dotted arrow is as described in FIG.
- FIG. 3 is a schematic view of one embodiment of the microchamber chip (1) used in the present invention viewed from directly above, and the pitch (50) indicates the distance between the centers of two adjacent microchambers (6). Show. The points in the figure represent a microchamber similar to the microchamber (6). Note that the direction indicated by the dotted arrow is as described in FIG. FIG.
- FIG. 4 is a schematic view in which a specific embodiment of the cell deployment device (10) according to the present invention is viewed from directly above.
- FIG. 4 (A) and FIG. 4 (A) are cross-sectional schematic views taken along the line A ′. B) is shown.
- the direction indicated by the dotted arrow is as described with reference to FIG. 1.
- the reservoir (60) (capacity is, for example, 500 ⁇ L) is disposed on the outlet (4), and the direction from the inlet (3)
- the flow-in cell suspension is discharged from the outlet (4) and temporarily stored in the reservoir (60).
- the ceiling portion and the side surface portion of the flow path forming frame (2) are formed from a flow path top plate (2a) and a flow path seal (2b), respectively.
- FIG. 5 shows an image of the surface of the microchamber chip after introducing the cell suspension into the flow path of the device for cell deployment in Example 1 and allowing it to stand for 5 minutes.
- FIG. 1 shows an image of the surface of a microchamber chip after intermittent liquid feeding in Example 1.
- FIG. 5C shows an arrangement pattern of the concave portions (110a) (holding portions (110)) of the plate (100) of the chemical microdevice described in Japanese Patent Application Laid-Open No. 2004-212048 (Patent Document 2).
- (D) shows the arrangement pattern of the microchamber (6) of the microchamber chip (1) used in Example 1, and each arrow represents the path and direction in which the cells contained in the cell suspension move. .
- FIG. 6 shows the formation of a large number of fine recesses (110a) as holding parts (110) for holding a sample on the surface of a chemical microdevice plate (100) described in JP 2004-212048 (Patent Document 2).
- FIG. 2 is a schematic plan view showing the state (see FIG. 1 of the publication, with the reference numerals changed).
- FIG. 7 is FIG. 7 described in International Publication No. 2010/027003 (Patent Document 1).
- Patent Document 1 In the top image of each of the microarray chips A to C having microchambers having different inner diameters and depths, A state in which red blood cells are stored is shown.
- the "cell deployment device” (10) has a “microchamber” (6) that can store and hold cells on the surface.
- the opening of the microchamber (6) is projected perpendicularly to the ⁇ longitudinal width of the microchamber chip (1) '' (20)
- the opening of the microchamber (6) is When projected perpendicularly to the vertical width (20) of the microchamber chip (1) (not the vertical width of the flow path), the projected opening of the microchamber (6) is in relation to the vertical width (20).
- the “void ratio”, which is the ratio of the sum of the voids (40), which is the length of the nonexistent portion, to the vertical width (20) is 5% or less, preferably 0%.
- the above “longitudinal width of the micro chamber chip (1)” (20) is defined as the width in the short direction of the micro chamber chip (1) as shown in FIG.
- the width in the longitudinal direction of the microchamber chip (1) is defined as the lateral width (30).
- the direction in which the cell suspension flows in the channel that is, the direction of the straight line connecting the inlet portion (3) and the outlet portion (4)
- the vertical width (20) of (1) is the width in the direction perpendicular to it.
- the device for cell development By using the device for cell development according to the present invention, a series of steps of developing a cell suspension on a microchamber chip, storing and holding cells in the microchamber, and staining and detecting specific rare cells are performed. It can also be automated.
- the microchamber chip (1) used in the present invention has one or more microchambers (6) capable of storing and holding one or more cells on its surface, and the porosity is preferably 5% or less, preferably Is 0%.
- the microchamber in the present invention refers to a concave fine hole (microwell) that can “store” and “hold” one or more cells, and has a bottom (that is, not a through hole). preferable.
- “storing” means that when a cell suspension is added to the surface of the microchamber chip for cell deployment according to the present invention, cells enter (accommodate) the microchamber, and “holding” means This means that the cells stored in the microchamber do not come out of the microchamber due to a staining solution or a washing solution added to the surface of the microchamber chip for cell development.
- FIG. 5 (C) shows a typical plate (chemical microdevice plate described in Japanese Patent Laid-Open No. 2004-212048 (Patent Document 2)) (100) in a concave portion (11a) (holding portion). (110)) shows the arrangement pattern, and when the cells contained in the cell suspension move along the path indicated by the arrow, the cells are stored in the recess (110a) when passing over the recess (110a). (Indicated by “ ⁇ ”), but the cells pass through the plate (indicated by “x”) if they do not pass over the recess (110a) at all. Note that the porosity of this plate is about 50%.
- FIG. 5 (D) shows a preferable arrangement pattern of the microchamber (6) according to the present invention, and the porosity of the microchamber chip having such an arrangement in the transverse direction of the channel is shown. Is 0% by eye.
- FIG. 5 (D) when the cells contained in the cell suspension move along the path indicated by the arrow, the cells are added on the microchamber (6) in any path. It can be seen that the possibility of being stored in the microchamber (6) is extremely high.
- the cells that can be held in each microchamber with respect to the total number of cells contained in the cell suspension It can be seen that the total number ratio (cell recovery rate) is extremely high.
- the pitch (50) of the microchamber (6) is preferably 1.5 times or more the diameter of the microchamber (6).
- the pitch is preferably the same as the diameter of the chamber (that is, the chambers are in contact with each other).
- the microchamber chip (1) is mass-produced using a mold, the chip is removed from the mold. The minimum pitch that can be removed is preferred.
- the diameter of the microchamber (6) is preferably 20 to 150 ⁇ m. When the diameter of the chamber exceeds 150 ⁇ m, the cell holding power of the chamber tends to be weakened, and when the diameter of the chamber is less than 20 ⁇ m, the cells may not be stored in the chamber.
- the depth of the microchamber (6) is preferably varied depending on the diameter of the microchamber (6), and those skilled in the art will be able to store about 10 to 15 cells per chamber.
- the thickness can be determined as appropriate.
- the depth of the microchamber (6) is 20 ⁇ m or more and 100 ⁇ m.
- the shape of the microchamber (6) is an inverted conical shape with a flat bottom in FIGS. 1 to 3 (the longitudinal section indicates a trapezoid), but the present invention is not limited to this embodiment.
- the bottom of the microchamber is typically flat, but may be curved.
- the material for the microchamber chip the same material as that of a conventionally known microplate can be used, and any material that can be molded using a mold is preferable.
- any material that can be molded using a mold is preferable.
- examples thereof include polymers such as siloxane [PDMS], polymethyl methacrylate [PMMA], and cyclic olefin copolymer [COC].
- the microchamber chip may be a combination of a plurality of materials in which a molded polymer is bonded to a substrate made of metal, glass, quartz glass, or the like.
- the manufacturing method of the microchamber chip (1) may be a method of forming using a mold having a convex portion corresponding to the shape of the microchamber (6) on the surface of the substrate, such as the polymer, metal, glass, etc.
- a method of forming a micro chamber by directly processing for example, fine processing by lithography, excavation processing, LIGA process, etc.
- a manufacturing method of forming using a mold is preferable.
- the microchamber chip (1) can be subjected to a surface treatment if necessary.
- the surface treatment include plasma treatment (oxygen plasma treatment, etc.), corona discharge treatment, treatment with a hydrophilic polymer, protein, lipid, and the like, but the present invention is not limited thereto.
- the flow path (5) formed in the cell deployment device (10) according to the present invention has a microchamber chip (1) surface as a bottom surface, for example, as shown in FIG.
- the flow path forming frame (2) provided integrally with (1) forms the side surface and the ceiling, and flows from the inlet (3) provided in the flow path forming frame (2).
- the cell suspension is allowed to flow in the direction indicated by the dotted arrow, and then the cell suspension flows out from the outlet (4) provided in the flow path forming frame (2).
- the flow path (5) has, for example, as shown in FIG. 1 (B), the bottom surface is the surface of the microchamber chip (1) and other members (in FIG. 1 (B), the flow path forming frame (2) ) May be formed.
- members other than the microchamber chip (1) are arranged in the short direction of the microchamber chip (1) in order to prevent cell loss due to flowing down the area without the microchamber (1). It is desirable not to form the bottom surface.
- microchamber chip (1) and the flow path forming frame (2) are integrated with each other, they can be detached from each other. It is also possible to remove only the ceiling (flow path top plate), leaving only the side surface from (2).
- the height (7) of the flow path (5) that is, the distance from the surface of the microchamber chip (1) other than the surface of the microchamber (6) to the ceiling (hereinafter also referred to as “ceiling height”). Is preferably 50 to 500 ⁇ m.
- the height of the ceiling is within the above range, it is easy to move cells attached to the surface of the microchamber chip (1) other than the surface of the microchamber (6) by the force of water flow, and the cells are microscopic. It is preferable because the time until it settles on the surface of the chamber chip (1) can be shortened, and the cells in the flow path are less likely to be clogged, so that the cells are smoothly developed.
- Examples of the material of the flow path forming frame (2) include the same materials as those of the micro chamber chip (1) as described above, and it is preferable to use the same material as the micro chamber chip (1). Further, the same surface treatment as that of the microchamber chip (1) as described above may be performed.
- the flow path (5) is, for example, as shown in FIG. 4 (B), between the flow path top plate (2a) and the microchamber chip (1), a silicon sheet having adhesiveness on both sides, that is, a flow.
- the channel forming frame (2) can also be formed by sandwiching the channel seal (2b).
- the cell suspension may contain rare cells, for example, blood such as human, lymph fluid, tissue fluid, body cavity fluid, etc., and may be appropriately diluted with a diluent or the like.
- the cell suspension is not limited to those derived from living organisms, and may be a cell dispersion prepared by suspending cells artificially for testing and research.
- rare cells examples include cancer cells.
- the rare cells may be CTC [circulating tumor cells or circulating cancer cells].
- the diameter of various cells contained in such a cell suspension is preferably 10 to 100 ⁇ m.
- a rare cell detection method can be used to change a cell suspension from a cell suspension to a cell suspension.
- a method for detecting a rare cell that may be contained by introducing a cell suspension into a flow path (5) from an inlet (3) of a cell deployment device (10), and A step of spreading on the surface of the chip (1); and a step of storing the developed cells in the microchamber (6), typically including the following steps.
- physiological saline such as PBS [phosphate buffered saline] from the inlet (3) of the cell deployment device (10) (preferably the same solvent as the cell suspension used in step (b)
- the cross-sectional area of the channel is 1 to 1,000 mm 2
- the flow rate is 1 to 1,000 ⁇ L / sec.
- the flow rate per second should be adjusted so that the flow rate is 1 mm / sec or less.
- the liquid is fed from the inlet (3) and allowed to stand for 1 to 30 seconds (for example, 10 seconds).
- This step is preferably repeated twice or more, more preferably 10 times. In this step, when liquid feeding and standing are repeated, this liquid feeding is particularly referred to as “intermittent liquid feeding” in this specification.
- a washing liquid of introducing the washing liquid from the inlet part (3) and discharging it from the outlet part (4) at least once.
- the cells are held in the microchamber (6), it is difficult to be discharged from the outlet (4) together with the staining solution and the washing solution, whereas the microchamber (6) is not held in the microchamber (6).
- the cells attached to the surface other than) are easily discharged from the outlet part (4) together with the staining solution and the washing solution (this is referred to as “loss”).
- Example 1 A microchamber chip (made of polystyrene and molded using a predetermined mold) in which the microchamber (6) was arranged as shown in FIG. 5 (D) was used for a cell deployment device.
- the diameter of the microchamber of this microchamber chip was 100 ⁇ m
- the depth of the microchamber was 50 ⁇ m
- the shape of the microchamber was an inverted conical shape with a flat bottom.
- the pitch representing the distance between the centers of adjacent microchambers was 200 ⁇ m
- the porosity was 0%.
- the flow path of the cell development device was filled with PBS containing 3% by weight of bovine serum albumin [BSA] (hereinafter also referred to as “PBS containing 3% BSA”).
- PBS containing 3% BSA bovine serum albumin
- FIG. 1 An enlarged image of the microchamber chip surface at that time is shown in FIG.
- the cells were dispersed and settled inside and outside the microchamber. Thereafter, intermittent liquid feeding (1 ⁇ L of 3% BSA-containing PBS was fed at a flow rate of 0.1 mL / min and then allowed to stand for 5 seconds) was performed 10 times.
- FIG. 5 (B) Magnified image of the microchamber surface at this time is shown in FIG. 5 (B). Most of the cells scattered outside the microchamber before intermittent feeding could be stored in the microchamber. When the total number of cells that flowed into the cell expansion device was taken as 100%, cells corresponding to 98% could be stored in the microchamber. That is, the cell recovery rate was 98%.
- Example 2 In Example 1, the cell recovery rate was determined in the same manner as in Example 1 except that the porosity of the microchamber chip was changed to 1% and 5%, respectively. The intermittent liquid feeding was further performed 10 times (20 times in total), and the cell recovery rate was determined. The results are shown in Table 1.
- Example 1 the cell recovery rate was determined in the same manner as in Example 1 except that the porosity of the microchamber chip was changed to 10%. The results are shown in Table 1.
- Example 2 the cell recovery rate was determined in the same manner as in Example 1 except that the plate shown in FIG. 6 having a porosity of 75% was used. As a result, the recovery rate of cells at the end of 10 intermittent liquid feedings was 26%.
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Abstract
Description
・細胞展開用デバイス
例えば図1(A)に示されるように、本発明に係る「細胞展開用デバイス」(10)は、細胞を格納、保持することができる「マイクロチャンバー」(6)を表面に有する「マイクロチャンバーチップ」(1)と;マイクロチャンバー(6)上に「流路」(5)が形成されるようにしてマイクロチャンバーチップ(1)と一体化して設けられた「流路形成枠体」(2)と;細胞懸濁液を流路(5)に流入させるために流路形成枠体(2)に設けられた「入口部」(3)と;入口部(3)から流路(5)に流入された細胞懸濁液を流路(5)から流出させるために流路形成枠体(2)に設けられた「出口部」(4)と;を少なくとも有し、図2に示すように、マイクロチャンバー(6)の開口部を「マイクロチャンバーチップ(1)の縦幅」(20)に垂直に投影したとき、マイクロチャンバー(6)の開口部を、マイクロチャンバーチップ(1)の縦幅(20)(流路の縦幅ではない。)に垂直に投影したとき、投影されたマイクロチャンバー(6)の開口部が該縦幅(20)に対して存在しない部分の長さである空隙(40)の総和の、該縦幅(20)に対する割合である「空隙率」が5%以下、好ましくは0%である。 Hereinafter, the present invention will be described in detail.
-Cell deployment device For example, as shown in FIG. 1 (A), the "cell deployment device" (10) according to the present invention has a "microchamber" (6) that can store and hold cells on the surface. A “microchamber chip” (1) having a “channel formation” provided integrally with the microchamber chip (1) so that a “channel” (5) is formed on the microchamber (6). A “frame” (2); an “inlet part” (3) provided in the flow path forming frame (2) for allowing the cell suspension to flow into the flow path (5); And at least an “exit part” (4) provided in the flow path forming frame (2) for allowing the cell suspension flowing into the flow path (5) to flow out of the flow path (5), As shown in FIG. 2, when the opening of the microchamber (6) is projected perpendicularly to the `` longitudinal width of the microchamber chip (1) '' (20), the opening of the microchamber (6) is When projected perpendicularly to the vertical width (20) of the microchamber chip (1) (not the vertical width of the flow path), the projected opening of the microchamber (6) is in relation to the vertical width (20). The “void ratio”, which is the ratio of the sum of the voids (40), which is the length of the nonexistent portion, to the vertical width (20) is 5% or less, preferably 0%.
本発明で用いるマイクロチャンバーチップ(1)は、その表面に、一個以上の細胞を格納し、保持することができるマイクロチャンバー(6)を一個以上有し、その上記空隙率が5%以下、好ましくは0%である。 <Microchamber chip>
The microchamber chip (1) used in the present invention has one or more microchambers (6) capable of storing and holding one or more cells on its surface, and the porosity is preferably 5% or less, preferably Is 0%.
本発明に係る細胞展開用デバイス(10)の中に形成されている流路(5)は、例えば図1(A)に示すように、マイクロチャンバーチップ(1)表面を底面とし、マイクロチャンバーチップ(1)と一体化して設けられた流路形成枠体(2)が側面部と天井部を形成しており、流路形成枠体(2)に設けられた入口部(3)から流入された細胞懸濁液を、点線の矢印が示す方向に流すためのものであり、その後、細胞懸濁液は流路形成枠体(2)に設けられた出口部(4)から流出される。 <Flow path>
The flow path (5) formed in the cell deployment device (10) according to the present invention has a microchamber chip (1) surface as a bottom surface, for example, as shown in FIG. The flow path forming frame (2) provided integrally with (1) forms the side surface and the ceiling, and flows from the inlet (3) provided in the flow path forming frame (2). The cell suspension is allowed to flow in the direction indicated by the dotted arrow, and then the cell suspension flows out from the outlet (4) provided in the flow path forming frame (2).
細胞懸濁液は、希少細胞を含んでいる可能性がある、例えばヒトなどの血液、リンパ液、組織液、体腔液などであり、希釈液などで適宜希釈されていてもよい。また、細胞懸濁液は、生体由来のものに限定されず、試験・研究等のために人工的に細胞を懸濁させて調製した細胞の分散液であってもよい。 <Cell suspension>
The cell suspension may contain rare cells, for example, blood such as human, lymph fluid, tissue fluid, body cavity fluid, etc., and may be appropriately diluted with a diluent or the like. The cell suspension is not limited to those derived from living organisms, and may be a cell dispersion prepared by suspending cells artificially for testing and research.
本発明に係る希少細胞の検出方法は、例えば図1(A)によると、上記の細胞展開用デバイス(10)を用いて、細胞懸濁液から、細胞懸濁液に含有されている可能性のある希少細胞を検出する方法であって、細胞展開用デバイス(10)の入口部(3)から細胞懸濁液を流路(5)に導入し、細胞をマイクロチャンバーチップ(1)表面上に展開する工程;および、展開された細胞をマイクロチャンバー(6)内に格納する工程を少なくとも含み、典型的には、下記の工程を含む。 -Rare cell detection method According to the present invention, for example, according to FIG. 1 (A), a rare cell detection method can be used to change a cell suspension from a cell suspension to a cell suspension. A method for detecting a rare cell that may be contained by introducing a cell suspension into a flow path (5) from an inlet (3) of a cell deployment device (10), and A step of spreading on the surface of the chip (1); and a step of storing the developed cells in the microchamber (6), typically including the following steps.
工程(b):入口部(3)から細胞懸濁液を導入し、工程(a)で流路を満たしていた生理食塩水と置換するようにして、細胞懸濁液で流路(5)を満たす。入口部(3)から細胞懸濁液を導入するのと同時に、出口部(4)から生理食塩水が排出される。 Step (a): physiological saline such as PBS [phosphate buffered saline] from the inlet (3) of the cell deployment device (10) (preferably the same solvent as the cell suspension used in step (b)) Fill the flow path (5).
Step (b): The cell suspension is introduced from the inlet (3), and is replaced with the physiological saline filled in the channel in the step (a). Meet. Simultaneously with the introduction of the cell suspension from the inlet (3), physiological saline is discharged from the outlet (4).
図5(B)に示されるように、間欠送液することによって、工程(c)の、マイクロチャンバー(6)内に格納されずにマイクロチャンバー(6)表面以外の表面に付着された細胞が、マイクロチャンバー(6)内に格納されやすくなる。 Step (d): A physiological saline (preferably the same solvent as the cell suspension used in step (b)) of about 1/100 to 1/2 (for example, 1/50) of the volume of the cell suspension is flowed. When the cross-sectional area of the channel is 1 to 1,000 mm 2 , the flow rate is 1 to 1,000 μL / sec. (The flow rate per second should be adjusted so that the flow rate is 1 mm / sec or less. The liquid is fed from the inlet (3) and allowed to stand for 1 to 30 seconds (for example, 10 seconds). This step is preferably repeated twice or more, more preferably 10 times. In this step, when liquid feeding and standing are repeated, this liquid feeding is particularly referred to as “intermittent liquid feeding” in this specification.
As shown in FIG. 5 (B), the cells that are not stored in the microchamber (6) in the step (c) but are attached to the surface other than the surface of the microchamber (6) by intermittent liquid feeding. It becomes easy to be stored in the micro chamber (6).
[実施例1]
マイクロチャンバー(6)が図5(D)に示すように配置されたマイクロチャンバーチップ(ポリスチレン製であり、所定の金型を用いて成形した。)を細胞展開用デバイスに用いた。このマイクロチャンバーチップのマイクロチャンバーの直径は100μm、マイクロチャンバーの深さは50μmであり、マイクロチャンバーの形状は、底部が平坦な逆円錐形であった。隣接するマイクロチャンバー同士の中心間の距離を表わすピッチは200μmであり、また、空隙率は0%であった。マイクロチャンバーチップの縦幅および横幅はそれぞれ25mmおよび70mmであって、また、細胞展開用デバイスの流路の高さは100μm、流路の幅は15mm、流路の長さは40mmであった。すなわち、細胞展開用デバイスの流路の断面は1.5mm2であり、流路の容積は60mm3(=0.06mL)となる。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
[Example 1]
A microchamber chip (made of polystyrene and molded using a predetermined mold) in which the microchamber (6) was arranged as shown in FIG. 5 (D) was used for a cell deployment device. The diameter of the microchamber of this microchamber chip was 100 μm, the depth of the microchamber was 50 μm, and the shape of the microchamber was an inverted conical shape with a flat bottom. The pitch representing the distance between the centers of adjacent microchambers was 200 μm, and the porosity was 0%. The vertical and horizontal widths of the microchamber chip were 25 mm and 70 mm, respectively, and the height of the channel of the device for cell deployment was 100 μm, the width of the channel was 15 mm, and the length of the channel was 40 mm. That is, the cross section of the channel of the device for cell deployment is 1.5 mm 2 and the volume of the channel is 60 mm 3 (= 0.06 mL).
実施例1において、マイクロチャンバーチップの空隙率をそれぞれ1%および5%に変更した以外は実施例1と同様にして細胞の回収率を求めた。間欠送液をさらに10回(全20回)実施し、細胞の回収率を求めた。その結果を表1に示す。 [Example 2]
In Example 1, the cell recovery rate was determined in the same manner as in Example 1 except that the porosity of the microchamber chip was changed to 1% and 5%, respectively. The intermittent liquid feeding was further performed 10 times (20 times in total), and the cell recovery rate was determined. The results are shown in Table 1.
実施例1において、マイクロチャンバーチップの空隙率を10%に変更した以外は実施例1と同様にして細胞の回収率を求めた。その結果を表1に示す。 [Comparative Example 1]
In Example 1, the cell recovery rate was determined in the same manner as in Example 1 except that the porosity of the microchamber chip was changed to 10%. The results are shown in Table 1.
実施例1において、空隙率が75%である図6に示すプレートを用いた以外は実施例1と同様にして細胞の回収率を求めた。その結果、間欠送液10回終了時の細胞の回収率は26%であった。 [Comparative Example 2]
In Example 1, the cell recovery rate was determined in the same manner as in Example 1 except that the plate shown in FIG. 6 having a porosity of 75% was used. As a result, the recovery rate of cells at the end of 10 intermittent liquid feedings was 26%.
2 ・・・流路形成枠体
2a・・・流路天板
2b・・・流路シール
3 ・・・入口部
4 ・・・出口部
5 ・・・流路
6 ・・・マイクロチャンバー
7 ・・・流路(5)の高さ
10 ・・・細胞展開用デバイス
20 ・・・マイクロチャンバーチップ(1)の縦幅
30 ・・・マイクロチャンバーチップ(1)の横幅(マイクロチャンバーチップ(1)の長手方向)
40 ・・・空隙(マイクロチャンバー(6)の開口部をマイクロチャンバーチップ(1)の縦幅(20)に垂直に投影したとき、該縦幅(20)から、投影されたマイクロチャンバー(6)の開口部に相当する幅を差し引いた長さ)
50 ・・・ピッチ
60 ・・・リザーバー
100 ・・・プレート
110 ・・・保持部
110a・・・凹部 1 ・ ・ ・
40 ・ ・ ・ Void (when the opening of the micro chamber (6) is projected perpendicularly to the vertical width (20) of the micro chamber chip (1), the projected micro chamber (6) Minus the width corresponding to the opening)
50 ・ ・ ・
Claims (7)
- 細胞を格納、保持することができるマイクロチャンバー(6)を表面に有するマイクロチャンバーチップ(1)と、
マイクロチャンバー(6)上に流路(5)が形成されるようにしてマイクロチャンバーチップ(1)と一体化して設けられた流路形成枠体(2)と、
細胞懸濁液を流路(5)に流入させるために流路形成枠体(2)に設けられた入口部(3)と、
入口部(3)から流路(5)に流入された細胞懸濁液を流路(5)から流出させるために流路形成枠体(2)に設けられた出口部(4)とを少なくとも有する細胞展開用デバイス(10)であって、
マイクロチャンバー(6)の開口部をマイクロチャンバーチップ(1)の縦幅(20)に垂直に投影したとき、投影されたマイクロチャンバー(6)の開口部が該縦幅(20)に対して存在しない部分の長さである空隙(40)の総和の、該縦幅(20)に対する割合である空隙率が、5%以下である細胞展開用デバイス。 A microchamber chip (1) having a microchamber (6) on the surface capable of storing and holding cells; and
A flow path forming frame (2) provided integrally with the micro chamber chip (1) so that the flow path (5) is formed on the micro chamber (6);
An inlet part (3) provided in the flow path forming frame (2) for flowing the cell suspension into the flow path (5);
At least an outlet part (4) provided in the flow path forming frame (2) for allowing the cell suspension flowing into the flow path (5) from the inlet part (3) to flow out of the flow path (5). A cell deployment device (10) comprising:
When the opening of the microchamber (6) is projected perpendicularly to the vertical width (20) of the microchamber chip (1), the projected opening of the microchamber (6) exists with respect to the vertical width (20). A device for cell deployment, wherein the void ratio, which is the ratio of the sum of the voids (40), which is the length of the portion not to be treated, to the vertical width (20) is 5% or less. - 上記空隙率が0%である、請求項1に記載の細胞展開用デバイス。 The device for cell deployment according to claim 1, wherein the porosity is 0%.
- 上記マイクロチャンバー(6)のピッチ(50)が、該マイクロチャンバー(6)の直径の1.5倍以上である、請求項1または2に記載の細胞展開用デバイス。 The device for cell deployment according to claim 1 or 2, wherein the pitch (50) of the micro chamber (6) is 1.5 times or more the diameter of the micro chamber (6).
- 上記マイクロチャンバー(6)の直径が、20μm以上150μm以下である、請求項1~3のいずれか一項に記載の細胞展開用デバイス。 The device for cell deployment according to any one of claims 1 to 3, wherein the diameter of the micro chamber (6) is 20 µm or more and 150 µm or less.
- 上記流路(5)の高さ(7)が、50μm以上500μm以下である、請求項1~4のいずれか一項に記載の細胞展開用デバイス。 The cell deployment device according to any one of claims 1 to 4, wherein the flow path (5) has a height (7) of 50 µm or more and 500 µm or less.
- 請求項1~5のいずれか一項に記載の細胞展開用デバイス(10)を用いて、細胞懸濁液から、細胞懸濁液に含有されている可能性のある希少細胞を検出する方法であって、
細胞展開用デバイス(10)の入口部(3)から細胞懸濁液を流路(5)に導入し、細胞をマイクロチャンバーチップ(1)表面上に展開する工程;および、
展開された細胞をマイクロチャンバー(6)内に格納する工程
を含む希少細胞の検出方法。 A method for detecting rare cells that may be contained in a cell suspension from the cell suspension using the cell deployment device (10) according to any one of claims 1 to 5. There,
Introducing a cell suspension into the flow path (5) from the inlet (3) of the cell deployment device (10) and spreading the cells on the surface of the microchamber chip (1); and
A method for detecting a rare cell, comprising a step of storing developed cells in a microchamber (6). - 上記細胞懸濁液に含まれる細胞の直径が、10μm以上100μm以下である、請求項6に記載の希少細胞の検出方法。 7. The method for detecting a rare cell according to claim 6, wherein the diameter of the cells contained in the cell suspension is 10 μm or more and 100 μm or less.
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WO2015034011A1 (en) * | 2013-09-04 | 2015-03-12 | 独立行政法人産業技術総合研究所 | Microarray chip and cell analysis method using same |
WO2016043212A1 (en) * | 2014-09-19 | 2016-03-24 | コニカミノルタ株式会社 | Intracellular nucleic acid amplification preparation method, and cell analysis method utilizing same |
JP2016059346A (en) * | 2014-09-19 | 2016-04-25 | コニカミノルタ株式会社 | Cell development method and kit for it |
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Also Published As
Publication number | Publication date |
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US10584367B2 (en) | 2020-03-10 |
EP2871232A1 (en) | 2015-05-13 |
EP2871232A4 (en) | 2016-03-02 |
US20150337355A1 (en) | 2015-11-26 |
EP2871232B1 (en) | 2021-05-05 |
JP6218185B2 (en) | 2017-10-25 |
JPWO2014007190A1 (en) | 2016-06-02 |
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